Spin x tube

Proteolytic stability in human blood plasma was assessed as described previously (Mäde et al., 2014) with minor adaptations. 15 nmol of the N‐terminally TAMRA‐labelled compound were dissolved in 10 μL of distilled water and 1490 μL of human blood plasma (citrate stabilized). The mixture was incubated for 96 h at 37°C. At the indicated time points, samples (150 μL ) were withdrawn and equal volumes of ethanol and acetonitrile were added in order to precipitate plasma proteins. Samples were incubated at −20°C overnight and centrifuged at 12,000 g for 30 s. Supernatants were again incubated at −20°C for 20 min and afterwards filtered using SpinX tubes (0.22 μm, Costar). Filtrates were directly subjected to reverse phase high performance liquid chromatography (RP‐HPLC) using a Varitide RPC column (200 Å, 6 μm pore size, 4.6 × 250 mm) and a gradient of 20 to 60 % B in A for 30 min. Intact peptide was followed by fluorescence detection at an excitation wavelength of 525 nm and an emission wavelength of 572 nm. Degradation of each compound was independently performed twice (n = 2) and is presented as mean ± SEM (Supporting Information Figure S3)

Blood was collected from normal C57BL/6 mice and allowed to clot in a 1.7 ml tube and then serum was separated by centrifugation at 8000 rpm for 10 minutes. Serum was stored at −80°C after filtration in 0.22 µm Spin-X tube (Costar). Control or µMT mice were injected i.v. with 500 µL serum one day before dead cells were injected or acetaminophen treatment. To confirm the efficiency of serum transfer, at the time of peritoneal lavage or liver harvest serum was taken from each mouse and total IgG was measured by Mouse IgG Total ELISA kit according to the manufacturer's protocol (eBioscience).